The field of art to which this invention relates is processes for the isolation and purification of avermectin compounds, particularly processes for the extraction of avermectin compounds from a fermentation broth.
The avermectins are a group of broad spectrum macrolide anti-parasitic agents. They are produced by fermenting a strain of the microorganism Streptomyces avermitilis (e.g., ATCC 31267, 31271 or 31272 or mutants thereof) under aerobic conditions in an aqueous nutrient medium containing inorganic salts and assimilable sources of carbon and nitrogen. Typically, avermectins have been characterized by a nomenclature system that utilizes various letters and numbers to refer to specific substituents.
A subset of avermectins, the natural avermectins, includes compounds wherein the C.sub.5 position is substituted by methoxy (denoted by A) or hydroxy (denoted by B); the C.sub.25 position is substituted by secbutyl (denoted by a) or isopropyl (denoted by b); and the C.sub.23 position is substituted by hydroxy (denoted by 2) or attached to the C.sub.22 position by a double bond (denoted by 1). Thus, there are eight possible natural avermectins and specific compounds are referred to by using a combination of symbols such as B1a or B2b. In addition, a subset of the natural avermectins could be referred to, for example, by using the term B natural avermectins. The B natural avermectins refers to the four natural avermectins wherein the C.sub.5 position is substituted by hydroxy, the C.sub.25 position is substituted by either isopropyl or secbutyl and the C.sub.23 position is substituted by hydroxy or is connected to the C.sub.22 position by a double bond. The isolation and chemical structure of the eight natural avermectin compounds (sometimes referred to as the C-076 compounds) are described in detail in British Patent Specification No. 1573955.
There are a variety of processes for isolating avermectins from a growth medium and for the selective isolation of particular avermectins from a growth medium. For example, U.S. Pat. No. 4,423,211 describes a process wherein the pH of the fermentation broth is adjusted within the range of 1.5 to 6 with a mineral acid such as sulfuric, hydrochloric or nitric acid. Then, the broth is contacted with and agitated with an extractant such as toluene, and the avermectin components are dissolved into and taken up by the extractant. The solution comprising the avermectin and the extractant is then passed through a separator to strip off the extractant, and the remaining avermectin isolated by, for example, crystallization.
In addition, U.S. Pat. No. 5,077,398 describes a two step process in which B1 components are crystallized, from a concentrated water immiscible solvent extract of the avermectin containing broth, with an alcohol or hydrocarbon/alcohol mixture. B2 components are crystallized from the leftover extract (which remains after the B1 crystallization) subsequent to its supersaturation (e.g., by lowering the temperature) utilizing B2 seed crystals.
Although the above methods have advanced the art these methods still depend on the use of nonaqueous solvents and environmental concerns dictate the reduced use of solvents whenever possible. Thus, there is still a continuing search for new methods of isolating avermectins.